7-substituted mitomycin a



1969 MASANAO MATSUI ET AL .3,420,846

7-SUBSTITU'IED MITOMYCIN A Filed July 19. 1965 Sheet of s 1M 044d WM,

BY z q u/C M ATTORNEYQI 7, 1969 MASANAO MATSUI ET AL Y'SUBSTITUTEDMITOMYCIN A Filed July 19, 1965 Sheet g of 3 Jan. 7, 1969 MASANAO MATSUIET AL 3,420,846

7-SUBSTITUTED MITOMYCIN A 0 Filed July 19, 1965 Sheet 3 of 5 ZOOOI /G 8WWW/M INVENTORS BYVUMM 4 4 -4 M A' ITORNEYr United States Patent3,420,846 7-SUBSTITUTED MITOMYCIN A Masanao Matsui, Tokyo, Keizo Uzu,Shizuoka-ken, and Yasuhiro Yamada and Shigetoshi Wakaki, Tokyo, Japan,assignors to Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan, a corporation ofJapan Filed July 19, 1965, Ser. No. 472,813 Claims priority, applicationJapan, Aug. 25, 1964, 39/47,830 US. Cl. 260-3263 2 Claims Int. Cl. C07d27/54 ABSTRACT OF THE DISCLOSURE Compounds having the formula:

wherein X is selected from the group consisting of N-lysino, N-argininoand NHR R being any alkyl group having from 3 to 6 carbon atoms or aphenyl group and Y is selected from the group consisting of H, methyland benzoyl.

The present invention relates to new antibiotics, more particularly tonew derivatives of mitosane compounds.

In 1956, Hata et a1. isolated new antibiotics from the fermented brothof Streptomyces caespitosus and named the antibiotics mitomycin A and B.In 1958, Wakaki et al. isolated a third antibiotic from the same brothand named it mitomycin C. Since then it has been determined that thechemical composition of the three compounds is as follows:

H300 CHzOCONH2 OCHa H30 N 6 :NH

MitomycinA HaCO- CHaOCONHa I i 1130- I N O NCHs Mitomycin C 3,420,846Patented Jan. '7, 1969 "ice Generally, these compounds correspond to theformula CHzOCONHz wherein OCH; NH: OH OCH: OH; H

and are called mitosane compounds.

Mitomycin A, B and C are excellent antibiotics, but they have a defectin that they are toxic to human blood.

- In order to obtain less toxic mitosane compounds, some research hasbeen conducted even before the determination of the chemical compositionof mitomycin A, B and C; for example, monomethyl-mitomycin C orporifiromycin was synthesized by reacting mitomycin C and methyl iodidein the presence of potassium carbonate. After the determination of thechemical composition of mitomycin A, B and C, research to obtainderivatives has been actively conducted. For example, Belgian Patent624,559 provides some 6,7 and la-substituted derivatives of mitosanecompounds.

The present invention is concerned with novel mitosane derivatives ofreduced toxicity and of correspondingly enhanced utility as antibiotics,and to the preparation thereof.

The new antibiotics of the present invention are:

7 -anilino-9a-methoxymitosane 7 -n-butylamino-9a-methoxymitosane7-n-amylamino-9a-methoxymitos ane 7 -n-hexyl amino-9a-methoxymitosane7-N-lysino-9a-methoxymitosane 7 -N- at ginin0-9 a-methoxymitosane 1a-methyl-7- (n-propyl) amino-9a-methoxymitosane, and 1 a-b enzoy1-7-(n-propyl amino-9 a-methoxymitosane.

According to the present aspect, mitomycin A or a lasubstitutedderivative thereof is reacted with appropriate amino group-containingcompound, such as primary amine, secondary amine, amino acid, etc., andthe methoxy group at the 7-position of the starting compound is replacedby amino or substituted amino (cf. first aspect supra).

The reaction is carried out by dissolving the starting compound in anappropriate solvent, such as methanol, and adding an excess of thereactant amino compound. The reddish purple color of the startingsolution is changed to bluish purple, and this is accompanied by acorresponding change in the ultra-violet absorption spectra.

The following examples set forth illustrative, but not lirnitative,presently preferred embodiments of this aspect.

Example 1 7-anilino-9a-methoxymitosane 500 mg. (milligrams) of mitomycinA is dissolved in 20 ml. (milliliters) of methanol, followed by theaddition 4 followed by the addition of 150 mg. of L-lysine. Afterstanding for 1 hour, the color of the reaction mixture changes to bluishpurple. The reaction mixture is concentrated in vacuo and the resultingresidue is dissolved in 5 methanol, followed by the addition of ether asprecipitant. of 2 ml. of aniline. The reaction mixture is allowed to Th7-N-1y in -9a-methoxymitosane i obtained a stand for 2 hours at roomtemperature. The thus-obtained amorphous d Yi ld; 470 mg, green solutionis concentrated under reduced pressure, and Th i f -r d spectrum i Nujolis hown in FIG, 5, the obtained greenish paste is dissolved in ethylacetate and passed through a silica-gel column. Development and 10Example 6 elution are conducted by using the mixture of acetone andethyl acetate (1:3). The eluate is concentrated under7-N-arginino-9a-methoxymitosane reduced pressure and then petroleumether is added, as precipitant for the objective7-anilino-9a-methoxymito- Using 210 mg. of L-arginlne in place of 150mg. of L- sane. Yield: 430 mg. lysine in Example 5, 420 mg. of amorphouspowder of 7- Analysis.Calcd. for C H O N C, 61.45; H, 5.40;N-arginino-9a-methoxymitosane is produced from 350 N, 13.65. Found: C,61.20; H, 5.10; N, 13.26. mg. of mitomycin A. D

The infra-red spectrum in Nujol is shown in FIG. 1. The infra-redspectrum 1n NllJOl 1s shown 1n FIG. 6.

Example 2 Example 7 7 n butylammo3amethoxymltosan?1a-methyl-7-n-propylamino-9a-methoxymitosane 500 mg. of mitomycin A isdissolved in 5 ml. of methanol, and then an excess of n-butylamine isadded. After 50 mg. of la-methyl mitomycin A is dissolved in 10 ml.standing for 10 minutes, the color of the reaction mixture of methanol,followed by the addition of excess of n-prO- C ang s t luish purple fromreddish purple. Th miX- 25 plyamine. After 10 minutes the bluish purplereaction ture is concentrated in vacuo and the resulting residue mixtureis concentrated under reduced pressure. Crystalis dissolved in ethylacetate, the resulting solution being lization is conducted by ethylacetate. 40 mg. of bluish then passed through silica-gel column. Ethylacetate is purple needle crystals of la-methyl-7-n-propylamino9aused fordeveloping and eluting. The eluate is concenmethoxy-mitosane isobtained. M.P. 178 C. trated in vacuo, ether being added as precipitantfor the Anal sis.Calcd. for C H O N C, 58.45; H, 6.71; objective7-n-butylamino-9a-methoxymitosane. Yield: 410 N, 14.35. Found: C, 58.20;H, 6.50; N, 14.10. Infra-red spectrum Nujol is shown in FIG. 7.

Analysis.-Calcd. for C H O N C, 58.45; H, 6.71; N, 14.35. Found: C,58.20; H, 6.20; N, 14.05. Example 8 The infra-red spectrum in Nujol isshown in FIG. 2.

la-benzoyl-7-n-propylamino-9a-methoxy-mitosane Example 3 7 amy1amino 9amethoxymitosane 200 mg. of la-benzoyl mitomycin A is dissolved in 10 ml.of methanol, followed by the addition of 1 ml. of Usmg namylamme m Placef n'buty1amme E n-propylamine. The further treatment is conducted as in480 of 7n'amylanlmogafmethoxymltoSane Example 5. 145 mg. of bluishpurple needle crystals of lafrom 500 of mltomycmbenzoyl-7-n-propylamino-9a-methoxy-mitosane is obtain- Analyszs.Ca1cd.for C H O N C, 59.39; H, 6.98; N, 13.85. Found: C, 59.10; H, 6.58; N,Analysis.--Calcd. for C H O N C, 62.48; H, 5.28;

The 1nfra-red spectrum in NHJOl 1s shown in FIG. 3. N, 11 66 F d; C,62.10; H, 5.60; N, 11.40.

Example 4 Infra-red spectrum in Nujol is shown in FIG. 8. 1 9 th LD(mg./kg.-mouse) of the new derivatives of the Xy ammo" Oxyml Osanepresent aspect are shown in Table 1b and the minimum Using n-hexylaminein place of n-butylamine in Exam- 50 inhibition concentrations are shownin Table 2. ple 2, 490 mg. of 7-hexylamino-9a-methoxymitosane isproduced from 500 mg. of mitomycin A. TABLE 1 Analysis.-Calcd. for C H ON C 60.27- H 7.23' LD i I I 9 a a 50 mg./ N, Found' C: 60013 7:1O: N:7-anilino-Qa-methoxymitogane 375 The Infra-red spectrum In Nuiol s Shown1 H6 7-n-butylamino-9a-methoxymitosane 1875 E l 57-n-amylamino-9a-methoxymitosane 18.45 7 N1 9 th7-n-hexylamin0-9a-methoxymitosane 37.5 Oxyml Osane7-N-lysin0-9a-methoxymitosane 225.0 350 mg. of mitomycin A is dissolvedin 40 ml. of water, 7-N-arginino-9a-meth0xymit0sane 300 TABLE 2.MINIMUMINHIBITING CONCENTRATION (meg/ml.)

7-n buty1- 7-n-pr0pyl- 7-n-hexyl- 7-anilino- 7-N-lysino-7-n-argininoamino-9aamino-Qaaminoa- Qa-methoxy- Qa-methoxy-Qa-methoxymethoxymethoxymethoxyrm'tosane mitosane mitosane mitosanemitosane mitosane Staphylococcus aureus 209P 0. 094 0. 0. 78 0. 39 6. 2525 Sarcinalutea PcI 1001 0. 195 0. 39 1. 56 0. 048 6. 25 3.12 Bacillussubtilis ATCC 6633. 0. 097 0.195 0.78 0. 04s 6. 25 25 Salmonella typhi379 1 12.5 12.5 750 3.12 50 50 Shigella flezneri Za 3196 12. 5 12. 5 750a. 12 25 50 K L p 0/10 25 6.25 50 1.56 25 25 Proteus X 19 50 12.5 5012.5 50 50 Eschericha coli K-12 25 6. 25 50 12.5 25 50 Pseudomonasaeruginosa 35 50 12.5 50 12.5 50 50 Vibrio comma 62 0. 39 0.097 1.56 0.024 0.39 3.12 Mycobacterium tuberculosis 607 3.12 0.39 1.56 0.195 1. 5612.5 Streptococcus hamemolyticus 68. 0. 98 0. 78 3.12 0. 024 3. 12 12.5Streptococcus faecalis 5 0.097 0. 195 0.78 0. 048 6. 25 12.5 Diplococcuspneumoniae 119 0. 48 0. 048 0. 195 0. 024 0. 195 0.39 Corynebacteriumdiphtheriae 92. 0. 39 0. 39 12.5 0. 048 12. 5 1. 56

What is claimed is: 1. 7-N-1ysino-9a-methoxymitosane. 2.7-N-arginino-9a-methoxymitosane.

References Cited UNITED STATES PATENTS FOREIGN PATENTS 9/ 1964 Belgium.

6 OTHER REFERENCES Theilheimer, Synthetic Methods of Organic Chemistry:vol. 4, Abstract Nos. 345-348 pp. 123-4 OD 265T4 (1950).

Webb et a1.: J. Am. Chem. Soc., vol. 84, pp. 3185- 3187 (1962).

NICHOLAS S. RIZZO, Primary Examiner.

10 J. A. NARCAVAGE, Assistant Examiner.

U.S. C1. X.R. 424-274

